Escherichia coli heat-labile enterotoxin(LT) is a useful model for analyzing the structure-function relationship of ADP-ribosylating, binary toxins involved in diarrheal disease. Importantly, both LT and cholera toxin are known to be potent mucosal adjuvants. Analyusis of these toxins may lead to the development of non-toxic mutant analogs that may be useful vaccine constituents and safe reagents that potentiate mucosal immune responses. Our laboratory has idenitified several residues in the enzymatically active A subunit of LT that serve important enzymatic functions, including Glutamic acid 112 and Arginine 192. We have constructed and purified mutant versions of the intact LT containing substitutions at these and other positions to yield effectively non-toxic molecules. These mutant analogs have been used in a collaborative study to assess their efficacy inpromoting protective immune responses against Helicobacter felis in a murine model after oral immunization. Initial results reveal that even those mutants with double substitutions and lacking detectable enzymatic or toxic activity are capable of significantly enhancing protective responses in mice immunized with Helicobacter extracts and challenged orally. Additional experiments involving immunization (intranasal and oral) against other mucosal pathogens are in progress. Another area of work has focused on the mechanism(s) of toxin internalization and intracellular trafficking. While accumulating evidence suggests that retrograde transport from the Golgi to th endoplasmic reticulum is involved in intoxication, our recent evidence indicates that alternative pathways exist that can be enhanced by physical disruption of the Golgi and/or inhibiton of microtubule function in certain cell types suggesting that alternative mechanisms of trafficking are involved. The role of A subunit sequences in intracellular targeting is under investigation using genetic fusions to green fluorescent protein (GFP). Initial results indicate that the intact A subunit is preferentially targeted to the Golgi complex in transfected cells. Current and future efforts will focus on identifying the regions or structures of the A subunit that are responsible for this phenotype and what functional role the Golgi localization plays in toxic or adjuvant activity.